Light-Controlled Biocatalysis by Unspecific Peroxygenases with Genetically Encoded Photosensitizers.

Abstract

Fungal unspecific peroxygenases (UPOs) have gained substantial attention for their versatile oxyfunctionalization chemistry paired with impressive catalytic capabilities. A major drawback, however, remains their sensitivity towards their co-substrate hydrogen peroxide, necessitating the use of smart in situ hydrogen peroxide generation methods to enable efficient catalysis setups. Within this report, we introduce flavin-containing protein photosensitizers as a new general tool for light-controlled in situ hydrogen peroxide production. The use of light as an abundant, inexpensive, and switchable induction agent enables efficient spatial control of enzymatic activity. By genetically fusing FbFPs and UPOs, we have created two virtually self-sufficient photo-enzymes (PhotUPO), which have been thoroughly characterized. Subsequent testing of a versatile substrate panel with the two divergent PhotUPOs revealed two stereoselective conversions. The catalytic performance of the fusion protein was optimized through enzyme and substrate loading variation, enabling up to 24300 turnover numbers (TONs) for the sulfoxidation of methyl phenyl sulfide. The PhotUPO concept was upscaled to a 100 mg substrate preparative scale, enabling the extraction of enantiopure alcohol products.